Abstract

A series of novel 4-(3,5-dimethyl-1H-pyrazol-1-yl)-2-substituted phenyl/Heterocyclic Thieno[2,3-d] Pyrimidine (8 a-j) derivatives were synthesized by a facile Five-step procedure that afforded advantages of mild reaction conditions, simple protocol and good yields. The structures of the final compounds were confirmed by IR, NMR, EI-MS. The final compounds were screened for their anti-bacterial activity against Bacillus subtilis and Staphylocouccus aureus from Gram positive group of bacteria and Escherichia coli and Klebsiella pneumonia from Gram negative group of bacteria and antifungal activity against Candida albicans and Aspergillus flavus. Anti-bacterial and anti-fungal activities were Evaluated and compared with the standard drugs Such as Amoxicillin and Ketoconazole. From anti-bacterial and antifungal activity screening results, it has been observed that compounds 8i, 8h, 8e and 8j possess good activity.

Keywords

Introduction

Pyrimidine has always been a unique interesting Heterocyclic
moiety for the medicinal chemists; an exhaustive research has been
done on the pyrimidines that led to the discovery and introduction of
several drugs into the market. From the standpoint of Biological activity,
fused hetero aromatic systems are often of much greater interest than
the constituent monocyclic compounds. The appearance of qualitatively
new properties of an annulated molecule, enlargement of the possibility
of varying pharmacophore groups in different positions of the molecule
and the ability of the latter to interact with a wider spectrum of receptors
adopting various conformations are apparently of crucial importance.
In addition, the structure of the molecule can be varied by annealing at
different positions of individual Heterocyclic fragments.

Fused Pyrimidines have also been attracted a considerable
interest in medicinal chemistry research due to their versatility and
a broad bioactive potential. Thieno pyrimidine is among those fused
pyrimidines found to have a wide variety of pharmacological and
biological applications. Since last four decades research has been
focused on the design and synthesis of novel thieno pyrimidines as
medicinal agents, a large number of reports have been documented
on thieno pyrimidines as they found to exhibit a variety of biological
activities such as antimicrobial, anti-inflammatory, bronchodilatory
activity, inhibition of Phospodiesterases, tyrosine kinase and VEGFR
kinase. It is evident that purine as an endogeneous scaffold plays an
important biochemical role in variety of regular physiological functions
such as respiration, inflammation, cell proliferation and so forth. As
a bio isoster to Purines, thieno[2,3-d] pyrimidines were also found to
exhibit numerous biological activities probably due to the interaction
with various physiological elements.

Thieno Pyrimidine is a bi cyclic heterocyclic compound consists of
a five membered thiophene ring is fused to a six-membered hetero cylic
ring with two nitrogen atoms. The fusion may occur in three different
orientations that results in three important types of thieno pyrimidines namely; Thieno[2,3-d]Pyrimidine (a), thieno[3,2-d]Pyrimidine (b) and
thieno[3,4-d]pyrimidine (c). Most of the isomeric thienopyrimidines
occur as colored amorphous form, some exists as crystalline form.

Synthetic approaches for the construction of a number of thieno
Pyrimidines are well established. There exists three possible types of
fusion of thiophene to pyrimidines ring results in corresponding
isomeric thienopyrimidines namely; thieno [2,3-d]pyrimidines (a),
thieno[3,4-d] pyrimidines (b) and thieno[3,2-d] pyrimidines (c).

Heterocycles containing the Thieno Pyrimidine moiety (Figure
1) are of interest because of their interesting pharmacological and
biological activities [1-6]. Thus, over the last two decades many thieno
Pyrimidines have been found to exhibit a variety of pronounced
activities, for example, as anti-inflammatory [3,7], anti-microbial [3,8],
antiviral [9] and analgesic [7,10] agents. Some Thieno Pyrimidine
derivatives showed good antitumor activity [11].

Figure 1: Structures of different isomers of Thieno Pyrimidine.

As a logical consequence of thiophene – phenyl isosterism, similarly
thieno pyrimidines can be considered as bio isosteres of quinazolines,
which are extensively described in scientific and patent literature as
displaying a plethora of biological activities. The synthesis of thieno
pyrimidine derivatives as potential surrogates for the quinazoline
core (Figure 2) structure has therefore, become a routine strategy in
modern drug design and development. Thieno pyrimidines as isosteres
of quinazolines are shown here.

Thienopyrimidines can also be considered as structural
analogues of five-membered heterocycles such as purines and
thiazolopyrimidines. As interesting anti-HIV activity was discovered
within the thiazolo[5,4-d]pyrimidine series, whereas the thiazolo[4,5d]
pyrimidines lack antiretroviral activity. The structures of purines and
thiazolo pyrimidines are shown in the following Figure 3.

Figure 3: Structures of purines and thiazolo pyrimidines.

Synthesis of thienopyrimidines

The building of thieno[2,3-d]pyrimidine moiety has been achieved
either by annulations of pyrimidine nucleus on the parent thiophene
ring or annulations of thiophene nucleus on the parent pyrimidine ring.
Also, they obtained from acyclic compounds.

Annulations of pyrimidine on thiophene ring

The simple approach to the formation of a new pyrimidine ring
involves introducing a one-carbon fragment between two suitable and
vicinal functional groups in thiophene ring.

Using thiophene having vicinal amino ester groups

Thiophene derivatives having vicinal amino ester groups are
considered a suitable synthon for the synthesis of thieno pyrimidines
via its interaction with various suitable reagents.

With urea and their derivatives: Methyl 3-aminothiophene-
2-carboxylate (1) was condensed with urea (2) at 190°C to yield
thieno[3,2-d] pyrimidin-2,4(1H,3H)-dione (3) in high yield (Figure 6)
[15].

Figure 6: Synthesis of thieno[3,2-d] pyrimidin-2,4(1H,3H)-dione (3).

Moreover, thieno[2,3-d]pyrimidines 4 and 5 (Figure 7) showed potent anticancer activity at low concenrations against most of the
used human tumor cell lines when compared to doxorubicin as potent
anticancer drug [16].

Figure 7: Structure of thieno[2,3-d]pyrimidines 4 and 5.

Reactions of Thienopyrimidines.

Reactions attributed to thiophene ring.

Reactions at thiophene carbons.

Electrophilic substitutions like halogenation, Vilsmeier
formylation, nitration and alkylation, were demonstrated in thieno[2,3-d]pyrimidines (I) and thieno[3,4-d]pyrimidines (III) (Figure 8) involved position 6 and equivalent position 7, respectively,
which is typical of thiophene itself and suggested a weak influence of
annelation with the pyrimidine ring. A different situation is observed
for electrophilic substitution in thieno[3,2-d]pyrimidines (II), where
the influence of annelation of the Pyrimidine ring is stronger than the
effect of orientation of the sulphur atom in the thiophene ring and,
consequently, the attack occurred at position 7.

Figure 8: Electrophilic substitution reaction of various Isomers of thieno[2,3-d]pyrimidine.

This work aimed to synthesize some new thieno[2,3-d]pyrimdine
derivatives starting with methyl 2-aminothiophene-3-carboxylate and
urea, to evaluate their Biological activities.

Encouraged by the diverse biological activities of Thieno [2,3- d]pyrimidine Heterocyclic compounds, it was decided to prepare
a new series of Thieno[2,3-d]pyrimidine Heterocyclic compounds.
Literature survey revealed that incorporation of different groups in
Thieno [2,3-d]pyrimidine Heterocyclic ring enhanced antibacterial
and antifungal activity. In the present communication 2-chloro-4-
hydrazinyl thieno[2,3-d] pyrimidine (4) was reacted with Acetyl
acetone (5) in Ethanol at Reflux Temperature to form Pyrazole Thieno
Pyrimidine[2,3-d] derivative (6), which was further reacted with
different types of boronic acids (7 a-j) under Suzuki reaction conditions to get target compounds (8a-8j) (Figure 13). The synthesis of the
compounds as per the following given below. The synthetic route was
depicted in (Figure 14).

Figure 14: A plausible mechanism pathway for the formation of pyrazole (6).

The structures of all synthesized compounds were assigned on the
basis of IR, Mass, 1H and 13C NMR spectral data analysis. Further these
compounds were subjected for antifungal and antibacterial activity.

Materials and Methods

In this Investigation chemicals were purchased from local dealer
with S.D fine make was used. Chemicals were 99% pure; purity has been
checked by thin layer chromatography and melting point. Conventional
method has been used for synthesis of thieno [2,3-d] Pyrimidine
derivatives. Stirring and reflux method were used for synthesis of Thieno [2,3-d] Pyrimidine derivatives 8 (a-j) respectively. The synthetic
route was depicted in Figure 13.

The title compounds 8(a-j) were synthesized in five sequential
steps using different reagents and reaction conditions, the 8(a-j) were
obtained in moderate yields. The structure was established by spectral
(IR, 1H -NMR, 13C-NMR and mass) data.

Experimental section

All reactions were carried out under argon in oven-dried glassware
with magnetic stirring. Unless otherwise noted, all materials were obtained
from commercial suppliers and were used without further purification.
All solvents were reagent grade. THF was distilled from sodium benzo
phenone ketyl and degassed thoroughly with dry argon directly before use. Unless otherwise noted, organic extracts were dried with anhydrous
Na2SO4, filtered through a fitted glass funnel, and concentrated with a
rotary evaporator (20-30 Torr). Flash chromatography was performed
with silica gel (200-300 mesh) by using the mobile phase indicated. The
NMR spectra were measured with a 400 MHz Bruker Avance spectrometer
at 400.1 and 100.6 MHz, for 1H for 13C, respectively, in CDCl3 solution
with tetra methyl silane as internal standard. Chemical shifts are given in ppm (δ) and are referenced to the residual proton resonances of the
solvents. Proton and carbon magnetic resonance spectra (1H NMR and 13C NMR ) were recorded using tetra methyl silane (TMS) in the solvent
of CDCl3-d1 or DMSO-d6 as the internal standard (1H NMR: TMS at 0.00
ppm, CDCl3 at 7.26 ppm, DMSO at 2.50 ppm; 13C NMR : CDCl3 at 77.16
ppm, DMSO at 40.00 ppm).

Synthesis

General procedure for synthesis of thieno[2,3-d]pyrimidine-2,4-
diol[compound (2)]:Methyl 2-aminothiophene-3-carboxylate (0. 1
mol, 15.7 g) and urea (0.5 mol, 30 g) were mixed with each other, and
the mixture was heated for two hours at 200°C. A clear, brown molten
mass was formed which solidified upon standing; the solid product
was dissolved in warm 1 N sodium hydroxide, and then acidified with
2 N Hydrochloric acid. The crystalline precipitate formed thereby
was collected by vacuum filtration and re-crystallized from Water,
yielding 72% (16.8 gms) of thieno[2,3-d]pyrimidine-2,4-diol, M.P.
300°C above.

General procedure for synthesis of 2,4-dichlorothieno[2,3-d]
pyrimidine [compound (3)]: A mixture consisting of (8.4 gm, 0.05 mol)
2,4-di hydroxy-thieno[2,3-d] Pyrimidine (2) (8.4 gm, 0.05 mol) and
100 ml. of phosphorus oxy chloride was refuxed for 10 hours, whereby a
clear solution was formed. Thereafter, the excess un reacted phosphorus
oxy chloride was evaporated in vacuo, the residual oil was poured into
ice water, and the aqueous mixture was extracted with chloroform. The
chloroform phase was isolated, washed with water until neutral, then
dried over Sodium sulfate, the chloroform was evaporated in vacuo,
and the solid residue was re crystallized from ethanol. 7.65 gm. (75% of
yield) of 2,4-dichloro thieno[2,3-d]pyrimidine, M.P. 161-162°C., were
obtained.

General procedure for synthesis of 2-chloro-4-
hydrazinylthieno[2,3-d]pyrimidine[compound (4)]: A mixture of
2,4-dichlorothieno[2,3-d]pyrimidine [compound (3)] (0.1 mol, 20.4
gms) in methanol was taken and cooled to 0°C-5°C in an ice bath. Tri
Ethyl amine (0.3 mol, 30.3 gms) was added to the cold reaction mixture
and then hydrazine hydrate (95% Purity) (0.15 mol, 8 gms) was added
slowly at 5°C-10°C. The reaction mass was allowed to stir at room
temperature for 3 hrs, after completion of starting compound, the excess
amount of methanol and Tri Ethyl amine was removed under vacuum.
The residue was washed with water, finally petroleum ether then they
obtain solid was filtered and Dried under vacuum (Figure 15).

General procedure for synthesis of 2-chloro-4-(3,5-dimethyl-1Hpyrazol-
1-yl)thieno[2,3-d]pyrimidine compound(6): To a mixture
of compound(4) (0.1 mol, 20 g) in methanol (50 ml), acetyl acetone
compound (5) (0.12 mol, 12 g) was added the reaction mixture was
refluxed for 2 hrs and then The obtained solid was filtered off, dried and
re-crystallized from Ethanol to give compound (6).

Antibacterial studies: The newly prepared compounds were
screened for their antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumonia and Escherichia coli (clinical isolate) bacterial strains by disc diffusion method. A standard
inoculums (1-2 × 107 c.f.u./ml 0.5 McFarland standards) were
introduced on to the surface of sterile agar plates, and a sterile glass
spreader was used for even distribution of the inoculums. The disks
measuring 6 mm in diameters were prepared from What man no. 1
filter paper and sterilized by dry heat at 140°C for 1 h. The sterile disks
previously soaked in a known concentration of the test compounds were
placed in nutrient agar medium. Solvent and growth controls were kept.
Amoxicillin (30 μg) was used as positive control and the disk poured
in DMSO was used as negative control and the test compounds were dissolved in DMSO at concentration of 100 and 50 μg/mL. The plates
were inverted and incubated for 24 h at 37°C. The susceptibility was
assessed on the basis of diameter of zone of inhibition against Grampositive
and Gram-negative strains of bacteria. Inhibition of zone of
measured and compared with controls. The bacterial zone of inhibition
values is given in Table 1. The order of activity was 8i>8h>8e>8j>8d >8f
>8g>>8a>8b>8c.

Zone of inhibition measure in mm

Synthesised Compounds

Gram positive

Gram negative

Bacillus subtilis

Staphylocouccus aureus

Klebsiella pneumonia

Escherichia coli

100 µg/mL

50 µg/mL

100 µg/mL

50 µg/mL

100 µg/mL

50 µg/mL

100 µg/mL

50 µg/mL

8a

7.5

3.5

8

7

9.5

7

10.5

7.5

8b

7

4.5

7

4.5

8.5

6.5

9

7

8c

6

3

7.5

5

8

6

9.5

6

8d

10

8

11.1

9.5

12

11

13.5

11

8e

11.5

9

12.5

11

14.5

11.5

15.5

12

8f

9.5

7

9.5

7.5

12

10

12.5

10.5

8g

8.5

6.5

9.0

6.5

10.15

8

11

8

8h

12.5

10

14.5

10.5

15

13.5

16.5

12.5

8i

13

10.5

15

11.5

16.5

14

17

13

8j

11

9.5

11.5

8.5

12.5

12

13

11.5

Amoxicillin

15.7

12.6

17.4

13

18

14.6

19.6

15.5

Control (DMSO)

---

---

----

-----

----

-----

-----

-----

Table 1: Anti-bacterial activity of compounds 8(a-j).

Antifungal studies: The newly prepared compounds were screened
for their antifungal activity against Candida albicans and Aspergillus flavus in DMSO by agar diffusion method. Sabourauds agar media
was prepared by dissolving peptone (1 g), D-glucose (4 g) and agar (2
g) in distilled water (100 ml) and adjusting ph 5.7. Normal saline was
used to make suspension of corresponding species. Twenty millilitres
of agar media was poured into each Petri dish. Excess of suspension
was decanted and the plates were dried by placing in an incubator at
37°C for 1 h using an agar punch, wells were made and each well was
labelled. A control was also prepared in triplicate and maintained at
37°C for 3-4 days. The fungal activity of each compound was compared
with Ketoconazole as a standard drug. Inhibition zone were measured
and compared with the controls. The fungal zone of inhibition values
is given in Table 2.

Zone of inhibition measure in mm

Synthesised Compounds

Candida albicans

Aspergillus flavus

100 µg/mL

50 µg/mL

100 µg/mL

50 µg/mL

8a

8.5

5

7.5

5.5

8b

8

5.5

7

3.5

8c

6.5

4.5

7

4

8d

11.5

6.5

9

6

8e

13

11.5

10.5

8

8f

11

9

10

9

8g

9.5

7.5

8

6.5

8h

14.5

12

12.5

9.5

8i

17.5

12.5

16

12

8j

12.5

8

10.5

10

Ketoconazole

21

16

18.5

14

Control (DMSO)

---

---

---

---

Table 2: Anti-fungal activity of compounds 8a-j.

Results and Discussion

Chemistry

The reaction sequences Employed for synthesis of title compounds
are shown in. In the present work, the starting thieno[2,3-d]pyrimidine-
2,4-diol(2) was prepared from methyl 2-amino thiophene-3-
carboxylate (1) and Urea according to synthetic procedure was prepared
according to synthetic procedure [19]. 2,4-dichlorothieno[2,3-d]
pyrimidine (3) was prepared according to synthetic procedure [20].
The 2-chloro-4-hydrazinylthieno[2,3-d] pyrimidine (4) was prepared
according to synthetic procedure [21], which on further treatment
with Acetyl acetone (5) in Ethanol to get 2-chloro-4-(3,5-dimethyl- 1H -pyrazol-1-yl)thieno[2,3-d]pyrimidine (6) according to synthetic
procedure [21], which were treated with different substituted phenyl
boronic acids and Heterocyclic boronic acids under Suzuki reaction
conditions to get Target Novel Thieno[2,3-d]Pyrimidine derivatives
(8a-j) according to synthetic procedure [22]. All compounds displayed
IR, 1H and 13C NMR and mass spectra consistent with the assigned
structures. 1H NMR and IR spectrum of compounds (8 a-j) showed
singlet at 2.3 ppm, 3.8 ppm are due to the aromatic methyl group
protons and Aromatic methoxy group protons. The most characteristic
IR absorption bands are at 1140 cm-1 (C-O-C), 760 cm-1 (C-Cl) and
1324 and 1552 cm-1 (N-O Stretching in Nitro group). The mass spectra
of all the final derivatives showed comparable molecular ion peak with
respect to molecular formula.

Anti-microbial studies

The newly synthesized compounds (8a-j) were screened for their
in-vitro anti-bacterial activity against Bacillus subtilis, Staphylocouccus aureus, Klebsiella pneumonia and Escherichia coli using Amoxicillin
as standard by disc diffusion method (zone of inhibition) [23,24].
The test compounds were dissolved in di methyl sulfoxide (DMSO)
at concentrations of 50 and 100 μg/mL.The antibacterial screening
revealed that all the tested compounds showed good inhibition against
various tested microbial strains compared to the standard drug. Along
with the synthesized compounds 8i, 8h, 8e and 8j were found to be
more active against tested bacterial strains as compared to the standard.
Compound 8f exhibited moderate antibacterial activity against all tested
bacterial stains. The activities exhibited by the synthesized compounds
were due to both pyrazole and different substituted phenyl and
Heterocyclic rings linked with Thieno[2,3-d] Pyrimidine as a Core ring.
The in-vitro antifungal activities for compounds 8a-8j were determined
by agar diffusion method [25]. The results indicate that, among the
tested compounds 8i, 8h, 8e and 8j were active against all tested fungal
strains. The enhanced activities are due to electron withdrawing groups
viz., -CF3 and nitro attached to thieno [2,3-d] pyrimidine ring. All
other compounds such as, pyrazole and phenyl ring with methyl and
methoxy groups in thieno [2,3-d] pyrimidine showed lesser antifungal
activity as compared with standard Ketoconazole. Tables 1 and 2 depict
the antimicrobial screening results of the final compounds.

Conclusion

The research study reports the successful synthesis and antimicrobial
activity of novel thieno[2,3-d] Pyrimidine as a core unit
containing Pyrazole and different Substituted Phenyl / Heterocyclic
derivatives. The anti-microbial activity study revealed that all the
tested compounds showed good antibacterial and antifungal activities
against pathogenic strains. The structure and biological activity
relationship of title compounds indicate that the presence of electron
withdrawing groups like –CF3 and -NO2 groups attached to the phenyl ring and thiophene, Indole, pyridine rings were responsible for good
antimicrobial activity and hence compounds 8i, 8h, 8e and 8j exhibited
more potent anti-microbial activity of all tested pathogenic strains.

Acknowledgements

Authors are thankful to our College Chairman S Venkata Rami Reddy (SVR)
Sir, and Principal Dr. P Mallikarjuna Reddy Sir for providing us required facilities
and motivation for completion of the Research work. We also extend our gratitude
towards Laxai Avanti Life Sciences Pvt Ltd., Hyderabad for providing us facilities of
IR Spectra, 1H NMR for characterization of Novel Synthesized compounds.